Journal: Cell Death Discovery

Article Title: HDAC11 promotes both NLRP3/caspase-1/GSDMD and caspase-3/GSDME pathways causing pyroptosis via ERG in vascular endothelial cells

doi: 10.1038/s41420-022-00906-9

Figure Lengend Snippet: A HUVECs were treated with different concentrations of disulfiram and NSA (1.25, 2.5, 5, 10, or 20 μmol/L) for 13 h.The cell viability was evaluated by MTT assay ( n = 6). B HUVECs were pretreated with disulfiram or NSA (5 μmol/L) for 1 h and then stimulated with TNF-α for 12 h. The representative photographs of double-fluorescent staining with PI (red) and Hoechst 33342 (blue), and the quantification of PI-positive cells, ×100. Scale bar indicates 200 μm. C The cellular supernatant LDH level was evaluated with a cytotoxicity detection LDH kit. D The levels of IL-1β, IL-6, and IL-18 in the cellular supernatant were detected by ELISA assay. E The mRNA expressions of IL-1β, IL-6, and MCP-1 were determined by quantitative real-time PCR. F , G HUVECs were transfected with GSDME siRNA or NC siRNA for 48 h, and then the protein and mRNA expressions of GSDME were determined by western blotting and quantitative real-time PCR. H HUVECs were pretreated with disulfiram or NSA for 1 h and then incubated with TNF-α for 12 h after their transfection with GSDME siRNA or NC siRNA. The representative photographs of double-fluorescent staining with PI (red) and Hoechst 33342 (blue), and the quantification of PI-positive cells, ×100. Scale bar indicates 200 μm. I The cellular supernatant LDH level was evaluated with a cytotoxicity detection LDH kit. J The levels of IL-1β, IL-6, and IL-18 in the cellular supernatant were detected by ELISA assay. K The mRNA expressions of IL-1β, IL-6, and MCP-1 were determined by quantitative real-time PCR. ** P < 0.01 vs. control group or NC siRNA group; # P < 0.05, ## P < 0.01 vs. TNF-α group or NC siRNA + TNF-α group; $ P < 0.05, $$ P < 0.01 vs. GSDME siRNA + TNF-α group. Results are expressed as mean ± SD ( n = 3).

Article Snippet: After 48 h, cells were pretreated with disulfiram or NSA (MedchemExpress, New Jersey, USA) for 1 h and then incubated with TNF-α for 12 h. Disulfiram and NSA were dissolved in DMSO.

Techniques: MTT Assay, Staining, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Incubation

Journal: PLoS ONE

Article Title: Targeting NPL4 via drug repositioning using disulfiram for the treatment of clear cell renal cell carcinoma

doi: 10.1371/journal.pone.0236119

Figure Lengend Snippet: (A) Cell proliferation was determined by XTT assays at 96 h after transfection with siNPL4 (A; * , P < 0.0001). (B) Wound healing (left) and matrigel invasion (right) assays after transfection with siNPL4 (* P < 0.0001). (C) Cell proliferation was determined by XTT assays at 96 h after treatment with disulfiram. (D) Representative image of immunofluorescence analysis in cells with or without disulfiram. Anti-NPL4 antibody was not used in negative control.

Article Snippet: At 72 or 96 h after addition of siRNA, disulfiram (stock solution DMSO, Tocris Bioscience, Bristol, UK), or sunitinib (stock solution DMSO, Biorbyt, Cambridge, UK), cell proliferation was determined by XTT assays using a Cell Proliferation kit II (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s instructions.

Techniques: Transfection, Immunofluorescence, Negative Control

Journal: PLoS ONE

Article Title: Targeting NPL4 via drug repositioning using disulfiram for the treatment of clear cell renal cell carcinoma

doi: 10.1371/journal.pone.0236119

Figure Lengend Snippet: (A) In vitro cell proliferation assays following treatment with disulfiram, sunitinib, or the combination of disulfiram and sunitinib after 72 h (* P < 0.001). (B) Wound healing and matrigel invasion assays after treatment with disulfiram. (* P < 0.0001). (C) In vivo cell proliferation assays; Tumor volumes were calculated in nude mice 69 days after subcutaneous injection of 786-o cells treated with vehicle, disulfiram, sunitinib, or the combination of disulfiram and sunitinib. Four groups were examined: vehicle, disulfiram (200 mg/kg, 5 times a week), sunitinib (40 mg/kg, 5 times a week), and a combination of disulfiram and sunitinib (n = 8 for vehicle, n = 9 for disulfiram group, n = 4 for the sunitinib and combination groups).

Article Snippet: At 72 or 96 h after addition of siRNA, disulfiram (stock solution DMSO, Tocris Bioscience, Bristol, UK), or sunitinib (stock solution DMSO, Biorbyt, Cambridge, UK), cell proliferation was determined by XTT assays using a Cell Proliferation kit II (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s instructions.

Techniques: In Vitro, In Vivo, Injection

Journal: PLoS ONE

Article Title: Targeting NPL4 via drug repositioning using disulfiram for the treatment of clear cell renal cell carcinoma

doi: 10.1371/journal.pone.0236119

Figure Lengend Snippet: (A) NPL4 protein expression levels in RCC cell lines treated with control, disulfiram, sunitinib, or the combination of disulfiram and sunitinib for 48 h were determined by western blotting analyses. (B) Apoptosis assays were performed using flow cytometry at 72 h after drug treatment (*, P < 0.0001). (C) Caspase-3/7 activity was measured by determining fluorescence intensity in RCC cells treated with control, disulfiram, sunitinib, or the combination of disulfiram and sunitinib for 48 h (*, P < 0.001; **, P < 0.0001; left). Representative images of caspase-3/7 activity in RCC cell lines (right).

Article Snippet: At 72 or 96 h after addition of siRNA, disulfiram (stock solution DMSO, Tocris Bioscience, Bristol, UK), or sunitinib (stock solution DMSO, Biorbyt, Cambridge, UK), cell proliferation was determined by XTT assays using a Cell Proliferation kit II (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s instructions.

Techniques: Expressing, Control, Western Blot, Flow Cytometry, Activity Assay, Fluorescence

Journal: PLoS ONE

Article Title: Targeting NPL4 via drug repositioning using disulfiram for the treatment of clear cell renal cell carcinoma

doi: 10.1371/journal.pone.0236119

Figure Lengend Snippet: Absolute quantification of proteins downregulated by disulfiram (A) or combination treatment with disulfiram and sunitinib (B). (C) AKR1B1 and PSAT1 protein expression levels in RCC cell lines treated with control, disulfiram, sunitinib, or the combination of disulfiram and sunitinib for 48 h were determined by western blotting analyses.

Article Snippet: At 72 or 96 h after addition of siRNA, disulfiram (stock solution DMSO, Tocris Bioscience, Bristol, UK), or sunitinib (stock solution DMSO, Biorbyt, Cambridge, UK), cell proliferation was determined by XTT assays using a Cell Proliferation kit II (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s instructions.

Techniques: Quantitative Proteomics, Expressing, Control, Western Blot

Journal: Magnetic resonance in medicine

Article Title: (13) C magnetic resonance spectroscopic imaging of hyperpolarized [1-(13) C, U-(2) H5 ] ethanol oxidation can be used to assess aldehyde dehydrogenase activity in vivo.

doi: 10.1002/mrm.25286

Figure Lengend Snippet: FIG. 1. a: Typical ethanol (EtOH) and acetate 13C signals acquired 20 s after i.v. injection of hyperpolarized [1-13C, U-2H5] ethanol into a mouse. The surface receive coil was placed over the liver. The inserts show a 10 vertical scale expansion of the acetate signal and a resolution- enhanced ethanol signal showing the splitting due to carbon–deuterium coupling. b: Time dependence of the ethanol and acetate signals fol- lowing ethanol injection. The amplitude of the acetate signal has been multiplied 10. c–e: The effect of disulfiram treatment on acetate signal intensity. The [1-13C] acetate (175–190 ppm; c) and [1-13C] acetalde- hyde (200–215 ppm; d) regions of the spectra are shown from animals treated with the indicated disulfiram concentrations (mg/kg body weight) 24 h prior to the MR experiment. e: Time dependence of the acetate signal intensity following disulfiram treatment at the indicated concentrations (mg/kg body weight). For control animals and animals treated with 600 mg/kg body weight disulfiram, the shaded area indica- tes6 standard deviation (n¼ 3).

Article Snippet: Disulfiram Dose-Response Experiments Animals were given 100 or 600 mg/kg body weight of disulfiram (LKT Laboratories, Inc., St. Paul, MN) as a suspension in 5% w/v of gum arabicum, by oral gavage (30).

Techniques: Injection, Control, Standard Deviation